The use of transgenic technology to introduce heterologous DNA into animals has been contemplated for the production of specific proteins or other substances of interest, such as proteins of pharmaceutical interest (Gordon et al., 1987, Biotechnology 5: 1183-1187; Wilmut et al., 1990, Theriogenology 33: 113-123). Transgenic animals can express exogenous proteins under conditions that offer high yield of the protein in an active form and can incorporate post-translational modifications such as glycosylation that are necessary for full functionality.
Historically, transgenic animals have been produced almost exclusively by microinjection of a fertilized egg. The pronuclei of fertilized eggs are microinjected in vitro with nucleic acid such as xenogeneic or allogeneic heterologous DNA or hybrid DNA molecules. The microinjected fertilized eggs are then transferred to the genital tract of a pseudopregnant female (see, for example, Krimpenfort et al., in U.S. Pat. Nos. 5,175,384, 5,434,340 and 5,591,669).
Systems that can function as protein bioreactors are reproductive systems which produce a hard shell egg such as the avian reproductive system. In avians the production of an egg begins with formation of the large yolk in the ovary. The unfertilized oocyte or ovum (i.e., germinal disc) is positioned on top of the yolk sac. Upon ovulation or release of the yolk from the ovary, it passes into the infundibulum of the oviduct where it is fertilized if sperm are present, and then moves into the magnum of the oviduct that is lined with tubular gland cells. These cells secrete the egg white proteins, including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin, into the lumen of the magnum from which they are deposited onto the avian embryo and yolk.
The avian oviduct (for example, a chicken oviduct) offers outstanding potential as a protein bioreactor because of high levels of protein production, the promise of proper folding and post-translation modification of the recombinant protein, the ease of product recovery and the shorter developmental period of chickens compared to other potential transgenic species.
The use of retroviruses has proven to be the only dependable method of producing transgenic avians (see, for example, U.S. Pat. No. 6,730,822, issued May 4, 2004. However, the use of retroviruses, poses certain limitations, including limitations to the size of the transgene. The use of microinjection would overcome certain of these limitations.
Production of transgenic chickens by cytoplasmic DNA injection have been described in Sang et al, Mol. Reprod. Dev., 1: 98-106 (1989) and Love et al, Biotechnology, 12: 60-63 (1994) incorporated herein by reference in their entireties. However, to date, the production of transgenic chickens by means of DNA microinjection has been both inefficient and time consuming and has produced inconsistent results and a lack of germ-line transmission of the injected DNA. The problems associated with transgenic avian production by microinjection are believed to be due, at least in part, to the delicate structure of the fertilized or unfertilized germinal disc and the lack of devices and methods of manipulating the germinal disc.
What is needed, therefore, are devices and methods that provide for a micropipette (e.g., needle or injection needle) to be placed accurately and rapidly to a germinal disc for delivery of a substance such as nucleic acid component to the germinal disc.